In the bacterial portion of the project, we have made further progress towards structural and mechanistic elucidations of the T-box riboswitch system, as well as targeting the T-box RNA with small molecule inhibitors. Using phylogenetic analysis and methods that we previously developed in the lab to transcribe and assemble functionally relevant T-box-tRNA complexes, we redefined the functional T-box antiterminator. We further found that the newly redefined T-box antiterminator is necessary and sufficient for specific binding with an uncharged tRNA. Combining X-ray crystallography, SAXS, and cryo-EM methods (in collaboration with Wah Chius lab), we have obtained consistent structural information that describe in detail how the T-box riboswitch recognize tRNA, detect aminoacylation, and switch conformations to direct downstream gene expression. This work may inform the development of antimicrobials that target the T-box RNA, and the design of artificial RNA devices that act as sensors and regulators of gene expression. In addition, working with the Constantinos Stathopoulos lab (University of Patras, Greece), we contributed to the characterization of interactions between mainstream antibiotics with a glycine-responsive T-box in Staphylococcus aureus in silico and in vitro. Structural modeling rationalizes the nuclease-probing data to suggest that several antibiotics bind strategic locations on the T-box stem I-tRNA structure, and thus can directly modulate the genetic outcome of the T-box regulon. This collaborative work is now published in Nucleic Acids Research. In the eukaryotic portion of the project, we have made further progress in structural and mechanistic elucidations of the Gcn2 system. Gcn2 senses and manages amino acid/serum starvation, UV irradiation, oxidative/osmotic/ER stress, etc, is essential for cellular survival under stress, and is a key player in several cancers and neurodegenerative diseases. Similar to T-boxes, this multi-domain protein directly engages tRNAs and evaluates their aminoacylation status to detect nutrient limitation, and couples this readout with the activation of its dormant kinase activity. To achieve these functions, Gcn2 incorporated a domain borrowed from the Histidyl-tRNA synthetase (HisRS), the enzyme responsible for charging histidine onto tRNA-his. This represents an interesting case of enzyme repurposing and adaptation. We have carried out SAXS and crystallographic analyses of the HisRS-like domain of Gcn2. We have obtained diffracting crystals of the protein domain and structural determination is ongoing. The new structural information, when available, will help illuminate how the HisRS parent enzyme evolves into the HisRS-like sensory domain of Gcn2, whilst retaining a similar overall architecture.